Protein kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Uncontrolled signaling has been implicated in a variety of disease conditions including inflammation, cancer, arteriosclerosis, and psoriasis. Reversible protein phosphorylation is the main strategy for controlling activities of eukaryotic cells. It is estimated that more than 1,000 of the 10,000 proteins active in a typical mammalian cell are phosphorylated. As is well known in the art, the high energy phosphate, which drives activation, is generally transferred from adenosine triphosphate molecules (ATP) to a particular protein by protein kinases and removed from that protein by protein phosphatases. Phosphorylation occurs in response to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.), cell cycle checkpoints, and environmental or nutritional stresses. The phosphorylation process is roughly analogous to turning on a molecular switch. When the switch goes on, the appropriate protein kinase activates a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor.
The kinases comprise the largest known protein group, a superfamily of enzymes with widely varied functions and specificities. They are usually named after their substrate, their regulatory molecules, or some aspect of a mutant phenotype. With regard to substrates, the protein kinases may be roughly divided into two groups: those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate serine or threonine residues (serine/threonine kinases, STK). A few protein kinases have dual specificity and phosphorylate threonine and tyrosine residues. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The primary structure of the kinase domains is conserved and can be further subdivided into 11 subdomains. The N-terminal of the kinase domain, which contains subdomains I-IV, generally folds into a lobe-like structure that binds and orients the ATP (or GTP) donor molecule. The C terminal of the kinase domain forms a larger lobe, which contains subdomains VI-XI, binds the protein substrate and carries out the transfer of the gamma phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue. Subdomain V spans the two lobes. Each of the 11 subdomains contains specific residues and motifs or patterns of amino acids that are characteristic of that subdomain and are highly conserved.
The kinases may be categorized into families by the different amino acid sequences (generally between 5 and 100 residues) located on either side of, or inserted into loops of, the kinase domain. These added amino acid sequences allow the regulation of each kinase as it recognizes and interacts with its target protein.
The presence or absence of a phosphate moiety modulates protein function in multiple ways. A common mechanism involves changes in the catalytic properties (Vmax and Km) of an enzyme, leading to its activation or inactivation.
A second widely recognized mechanism involves promoting protein-protein interactions. An example of this is the tyrosine autophosphorylation of the ligand-activated EGF receptor tyrosine kinase. This event triggers the high-affinity binding to the phosphotyrosine residue on the receptor's C-terminal intracellular domain to the SH2 motif of an adaptor molecule Grb2. Grb2, in turn, binds through its SH3 motif to a second adaptor molecule, such as SHC. The formation of this ternary complex activates the signaling events that are responsible for the biological effects of EGF. Serine and threonine phosphorylation events also have been recently recognized to exert their biological function through protein-protein interaction events that are mediated by the high-affinity binding of phosphoserine and phosphothreonine to the WW motifs present in a large variety of proteins.
A third important outcome of protein phosphorylation is changes in the subcellular localization of the substrate. As an example, nuclear import and export events in a large diversity of proteins are regulated by protein phosphorylation.
Many kinases are involved in regulatory cascades wherein their substrates may include other kinases whose activities are regulated by their phosphorylation state. Ultimately the activities of some downstream effectors are modulated by phosphorylation resulting from activation of such a pathway.
Myotonic dystrophy kinase-related Cdc42 binding kinases (MRCKs) are serine/threonine kinases. MRCKs have been implicated in the morphological activities of Cdc42 in non-neural cells and are suggest to be downstream effectors of Cdc42 in cytoskeletal reorganization. At least two types of MRCKs, MRCK alpha and MRCK beta, have been identified. MRCKs interact with the GTP-bound form of Cdc42 and, to a lesser extent, the GTP-bound form of Rac. The catalytic domain of MRCKs phosphorylates non-muscle myosin light chain 2 at serine 19. The phosphorylation is believed to be involved in myosin contractile activity and associated changes in the organization of actin microfilaments in intact cells.
MRCK alpha and Rho-binding kinase (ROK) alpha are believed to have contrasting roles in regulating neurite morphology. ROK alpha acts downstream of RhoA in inducing neurite retraction, while MRCK alpha acts downstream of Cdc42/Rac1 in promoting neurite outgrowth. The neurite outgrowth induced by either kinase-dead ROK alpha or nerve growth factor can be effectively blocked by a kinase-dead and p21-binding deficient MRCK alpha mutant. In addition, expression of kinase-dead MRCK alpha blocks Cdc42V12-dependent formation of focal complexes and peripheral microspikes. Microinjection of a plasmid encoding MRCK alpha results in actin and myosin reorganization.
MRCKs have multiple functional domains. These domains include three coiled-coil alpha-helix domains, a cysteine-rich motif resembling those of protein kinase C and n-chimaerin, and a Pleckstrin homology domain. Native MRCK kinases tend to form high-molecular-weight multimers. The intermolecular interactions among the three coiled-coil domains and the N-terminal region preceding the kinase domain in MRCK alpha are believed to be responsible for the multimerization.
MRCK alpha can be activated by the N-terminus-mediated dimerization. The dimerization leads to trans-autophosphorylation of MRCK kinases. In addition, MRCK alpha kinases can be negatively regulated through intramolecular interactions between the two distal coiled-coil domains. Deletion of these coiled-coil domains leads to a more active kinase, showing the negative autoregulatory role of these domains. The N-terminus-mediated dimerization and the intramolecular interaction between the distal coiled-coil domains are considered to be two mutually exclusive events, which regulate the catalytic state of MRCK kinases.